Production using a genetically modified microorganism is advantageous over the production using mammalian cells with respect to several points including the low production cost and high culture technique that has been accumulated as the fermentation technology. Recently, attention has been paid to a methylotrophic yeast belonging to the genus Pichia (e.g., Pichia pastoris) as an effective host for producing a heterologous protein [Cregg, J. M. et al., Bio/Technology 11, 905 (1993)]. Yeasts belonging to the genus Pichia have much larger secretary expression amounts than Saccharomyces cerevisiae, and culture techniques for them are established, so that they are very suitably used as yeasts for industrially producing human serum albumin and so on.
In case a microorganism is used as a host for producing a heterologous glycoprotein, however, it is impossible to add a sugar chain having the structure and composition same as human glycoprotein, and this is a problem. For asparagine-linked sugar chain of glycoprotein derived from mammalian cells including human cells, three types are known, i.e., complex type, hybrid type, and high-mannose type. On the other hand, in prokaryotes such as Escherichia coli, the addition per se of sugar chain does not occur.
For asparagine-linked sugar chains added in Saccharomyces cerevisiae, only the high mannose type is known. With respect to asparagine-linked sugar chains of glycoprotein in S. cerevisiae, ER core-like sugar chain (Man8GlcNAc2) common with that of mammal is added in the endoplasmic reticulum, and in the following process a large amount (30-150 molecules) of mannose is formed as sugar outer chain [Kukuruzinska, M. A. et al., Ann. Rev. Biochem. 56, 915 (1987)]. Therefore, high mannose-type sugar chains having been added to the glycoprotein derived from S. cerevisiae contain more amount of mannose than that of a mammalian cell, i.e., they contain mainly hyper-mannosylated sugar chains.
The α-1,3 linkage in mannose of the sugar chain of S. cerevisiae is not found in sugar chains of mammalian including human being. Therefore, it is considered that this structure might exhibit an antigenic property to human beings [Ballou. C. E., Methods Enzymol., 185, 440-470 (1990)]. In addition, the fact that sugar chains are involved in various in vivo roles such as the blood clearance, the maintenance of protein structural, the contribution to activity, and the localization [Takeuchi, Tanpakushitsu Kakusan Kouso, special issue “Glycoconjugate”, 37, 1713 (1992)] suggests that a heterologous protein having a high mannose-type sugar chain produced by using S. cerevisiae has a big problem in the aspect of function.
Recently, with respect to S. cerevisiae, OCH1 gene coding α-1,6-mannosyltransferase that is the key enzyme for the sugar outer chain mannose elongation was cloned [Nakayama, K., EMBO J. 11, 2511 (1992)]. It was reported that in Δoch1-mnn1-double mutant having mutations in both OCH1 gene and MNN1 gene coding a protein having a function of adding mannose to the core-like sugar chain with the α-1,3 linkage, only ER core-like sugar chain common to that of mammal is added [Jigami, Y, Tanpakushitsu Kakusan Kouso, 39, 657 (1994)].
It is known that a mannose-type sugar chain is added in yeasts belonging to the genus Pichia in a manner similar to that of S. cerevisiae, while it was shown that the mannose addition number of a yeast belonging to the genus Pichia is less than that of S. cerevisiae and that sugar chain of yeasts belonging to the genus Pichia does not contain the α-1,3 linkage that is considered to be highly antigenic to human beings [Trimble, R. B. et al., J. Biol. Chem. 266, 22807 (1991)]. In addition, a gene homologous to OCH1 gene of S. cerevisiae was cloned, and it was confirmed that control of the gene in yeast strain belonging to the genus Pichia suppresses the elongation of the sugar chain. Therefore, this strain is useful as a host for producing a heterologous glycoprotein having a sugar chain structure similar to the human-type one [Japan Patent Laid Open Hei 9-3097].
Although a technique has been developed which suppresses the elongation of the sugar chain and permits expressing a glycoprotein similar to the ER core-like sugar chain, there remains the following problem also in a yeast belonging to the genus Pichia: a heterologous glycoprotein has an antigenicity caused by an acidic sugar chain in case the heterologous glycoprotein produced by using a yeast as a host is administered to human beings as a medicine.
It is known that acidic sugar chain added by mannose-6-phosphate (Man-6-P) to a core-type sugar chain and/or sugar outer chain are/is formed in S. cerevisiae [Hernandez, L. M. et al. J. Biol. Chem. 264, 13648-13659 (1989)]. As FIG. 1 illustrates, mannose phosphate is added not only to the sugar outer chain but also to the core-like sugar chain in S. cerevisiae [Jigami, Y and Odani, T., Biochim. Biophys. Acta, 1426, 335-345 (1999)]. This sugar chain containing the phosphate group does not found in human-type sugar chains. Therefore, it is very likely that this has an antigenicity and it is considered that this can be a big problem in case of developing a medicine. With respect to S. cerevisiae, MNN4 gene and MNN6 gene have been cloned and analyzed as genes participating in the transfer of mannose phosphate [Odani, T. et al. Glycobiology 6, 805 (1996); Wang, X.-H., et al. J. Biol. Chem., 272, 18117 (1997)]. It was confirmed in vitro that the transfer of mannose phosphate to the core-like sugar chain or Man5GlcNAc2 depends on Mnn6p (protein encoded by MNN6). Therefore, it is assumed that the MNN6 gene codes the main body of mannose phosphate transferase.
In addition, with respect to MNN4 gene, the mannose phosphate-transferring activity is suppressed in mnn4 mutant, and phosphate content is increased by the overexpression. Therefore, MNN4 gene is considered to be a factor that positively controls Mnn6 protein. It was elucidated that the transfer of phosphate to a sugar chain is decreased in yeast whose MNN4 gene was disrupted by a genetic engineering technique [Japan Patent Laid Open Hei 9-266792]. Therefore, the yeast can be used for producing a glycoprotein having a reduced antigenicity to human beings. However, even if S. cerevisiae MNN4 gene-controlled strain is used, the acidic sugar chain in the core-like sugar chain cannot be sufficiently suppressed, with less than 30% of the whole sugar chain being an acidic sugar chain [Odani T. el al., Glycobiology 6, 805-810 (1996); Japan Patent Laid Open Hei 9-266792].
On the other hand, with respect to yeasts belonging to the genus Pichia, only a few reports have been published concerning the phosphorylated sugar chain of a heterologous protein so far. For example, in kringle 2 domain of the tissue plasminogen activator, a mannose phosphate group had been transferred to 20% of the sugar chain of Man10-14GlcNAc2 [Miele, R. G. et al. Biotechnol. Appl. Biochem. 26, 79 (1997)]. In addition, although mannose phosphate group was detected in the sugar chain of Man9-14GlcNAc2 of aspartic protease, a phosphorylated sugar chain was not detected in five other heterologous proteins investigated at the same time [Montesino, R. et al. Protein Exp. Purif. 14, 197 (1998)]. Therefore, with respect to a yeast belonging to the genus Pichia, the frequency of the transfer of mannose phosphate to a sugar chain might be low, but phosphorylated sugar chains are detected in some heterologous proteins produced thereby. Therefore, it would be desirable to suppress the addition of mannose phosphate to the sugar chain in case administered to human beings as a medicine.
Although the expression system using a yeast belonging to the genus Pichia as a host is effective for the industrial production because of its high productivity, the mechanism of the transfer of mannose phosphate to the sugar chain of a glycoprotein originally possessed or produced, as a heterologous protein, by a yeast belonging to the genus Pichia has been studied only insufficiently. Therefore, with respect to a yeast belonging to the genus Pichia, the mechanism of the transfer of mannose phosphate to a glycoprotein sugar chain is quite unknown, and it is an important subject from the viewpoint of avoiding the antigenicity in the development of a medicine for administering to human beings or mammalians to suppress the transfer of mannose phosphate in a glycoprotein-expressing system in which yeast belonging to the genus Pichia is used as a host.
Thus, the purposes of the present invention are to find a gene participating in the addition of mannose phosphate to the sugar chain of a glycoprotein derived from a yeast belonging to the genus Pichia and to provide a means for controlling the addition. Other purposes of the present invention are to provide a method for producing a protein whose acidic sugar chain was reduced using a yeast strain belonging to the genus Pichia in which the gene is controlled and to provide a glycoprotein produced by the method.
The applicants' zealous examinations for solving these problems resulted in the success of the cloning of the gene coding a protein that is originated from a yeast belonging to the genus Pichia and participates in the addition of mannose phosphate, the finding that the protein participates in the addition of mannose phosphate in an expression system using a yeast belonging to the genus Pichia as a host, and the confirmation that acidic sugar chain of the glycoprotein produced by using the gene-controlled strain is remarkably reduced, and consequently reached the completion of the present invention.